Summary: Metabolic alterations in cancer cells have profound effects in disease development and progression. In recent years, there has been increasing interest in potential roles of fatty acid ?-oxidation (FAO) in tumorigenesis. While several studies have demonstrated that FAO is crucial for bioenergetic support of cancer cell growth, there is emerging evidence for potential involvement of FAO in immune modulation. However, the exact role of this metabolic pathway in antitumor immunity induced by cancer immunotherapy remains largely unknown. The proposed research seeks to understand the metabolic process of FAO, centered on its rate-setting enzyme carnitine palmitoyltransferase 1A (CPT1A), as a key contributing factor to tumor-induced immune dysfunction that impedes cancer immunotherapy. Our preliminary observations suggest that abnormal elevation in FAO impairs the function of dendritic cells (DCs), which are crucial for initiation and maintenance of T cell-mediated antitumor immunity. Strikingly, selective ablation of CPT1A in DCs markedly improves immunotherapeutic potency against established, poorly immunogenic tumors. In this application, we will use unique genetic tools and molecular/immunological approaches to test the hypothesis that the CPT1A-depedent FAO pathway defines a tolerogenic phenotype of tumor-associated DCs and promotes immune suppression (e.g., PD-L1/2) in the tumor microenvironment. We will determine the impact of DC-intrinsic FAO on cancer immunotherapies, including vaccines or immune checkpoint inhibitors, as well as mobilization of antigen-specific cytotoxic T lymphocytes (CTLs). Using preclinical mouse models and patient- derived specimens, we will define the mechanisms underpinning the FAO-impaired antigen-presenting function of DCs in response to immunostimulatory agents. We will also, for the first time, link this metabolic pathway functionally to a negative feedback regulator of inflammatory signaling, which can dictate the immunogenicity of DCs and immune tolerance. Moreover, we will investigate the elevation of FAO in cancer cells as a novel determinant of their therapeutic resistance to CTLs. Lastly, using the FAO-blocking drugs already approved for treatment of non-cancerous diseases we will test the concept of metabolic intervention of the FAO pathway to revitalize immune functions and to sensitize cancer cells to CTLs. Successful completion of this project is expected to elucidate a previously unrecognized immunosuppressive mechanism involving hyperactive FAO that promotes immune tolerance in the tumor-bearing host. New insights into this metabolic pathway that operates in both cancer cells and tumor-associated DCs will advance our understanding of a sub-optimal response in the majority of patients undergoing cancer immunotherapies. Our finding may open up an entirely new avenue for improved cancer immunotherapies by reprograming abnormal lipid catabolism to reinvigorate immune defense against cancer.